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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 30 July 1996 To 23 May 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: UKEMS Sub-Committee on Guidelines for Mutagenicity Testing: Report, Part II revised (Supplementary Mutagenicity Tests: UKEMS recommended procedures, 1993)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. QA statement)
Type of assay:
unscheduled DNA synthesis

Test material

Constituent 1
Chemical structure
Reference substance name:
A mixture of: tetrasodium-phosphonoethane-1,2-dicarboxylate; hexasodium-phosphonobutane-1,2,3,4-tetracarboxylate
EC Number:
410-800-5
EC Name:
A mixture of: tetrasodium-phosphonoethane-1,2-dicarboxylate; hexasodium-phosphonobutane-1,2,3,4-tetracarboxylate
Cas Number:
143239-08-1
Molecular formula:
Constituent 1 (tetrasodium phosphonoethane-1,2-dicarboxylate): Hill formula: C4H3Na4O7P CAS formula: C4 H7 O7 P .4Na Constituent 2 (hexasodium-phosphonobutane-1,2,3,4-tetracarboxylate): Hill formula: C8H5Na6O11P CAS formula: C8 H11 O7 P .6Na
IUPAC Name:
Reaction mass of tetrasodium phosphonoethane-1,2-dicarboxylate and hexasodium-phosphonobutane-1,2,3,4-tetracarboxylate
Details on test material:
- Name of test material (as cited in study report): ITC 288
- Physical state: white solid
- Storage condition of test material: ambient temperature (< 25°C), over silica gel
Specific details on test material used for the study:
Purity of the test material is based on the phosphonate composition, with no direct measure of % sodium. ITC 288 is not the "Reaction mass of tetrasodium-phosphonoethane-1,2-dicarboxylate and hexasodium-phosphonobutane-1,2,3,4--tetracarboxylate" (EC 410-800-5) rather the substance "Reaction mass of trisodium-phosphonoethane-1,2-dicarboxylate and pentasodium-phosphonobutane-1,2,3,4--tetracarboxylate" (EC 701-079-0) as demonstrated by the manufacturing specs reported in Section 1.2, legal entity composition.

Test animals

Species:
rat
Strain:
CD-1
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK, Margate, Kent.
- Age at study initiation: 6-9 weeks old
- Weight at study initiation: 221-265 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: up to 3 in solid-floor polypropylene cages
- Diet : ad libitum
- Water : ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data


IN-LIFE DATES: no data

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no data
- Amount of vehicle (if gavage or dermal): 10 ml/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: For the purpose of this study the test material was freshly prepared as required as a solution at the appropriate concentrations in distilled water


Duration of treatment / exposure:
not applicable
Frequency of treatment:
a single oral dose
Post exposure period:
2 hours (experiment 2) and 16 hours (experiment 1)
Doses / concentrations
Remarks:
Doses / Concentrations:
700 and 2000 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
4 males per dose (test substance and positive control) + 6 males (vehicle control)
Control animals:
yes, concurrent vehicle
Positive control(s):
2-acetylaminofluorene (AAF) and N,N'-Dimethylhydrazine dihydrochloride (NDHC) were freshly prepared as required as a suspension at the appropriate concentration in arachis oil BP and in sterile distilled water respectively.
- Route of administration: oral
- Doses / concentrations: 50 mg/kg (AAF) and 20 mg/kg (NDHC)

Examinations

Tissues and cell types examined:
Hepatocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A range-finding toxicity study was performed to determine a suitable dose level for the main UDS study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): In the first experiment, two groups of 4 rats were dosed with test material at 700 and 2000 mg/kg. In addition, two further groups of rats were included in the experiment, one group (6 rats) was dosed with the vehicle alone (distilled water) and the second group (4 rats) was dosed with Acetylaminofluorine as a positive controls. All animals were perfused approximately 16 hours after dosing. For pratical reasons the whole experiment was performed in phases, with animals from each group treated in each phase. In the second experiment, the dose groups and group sizes were as for Experiment 1 except that the positive control was N,N'-dimethylhydrazine dihydrochloride and animals were perfused approximately 2 hours after dosing.

DETAILS OF SLIDE PREPARATION:
The cells were fixed in freshly prepared 1:3 acetic acid:methanol, three changes of fixative were used. Finally cells were washed with distilled water and the coverslips allowed to air dry and then mounted onto the ends of glass slides, cell side up, and left to dry overnight in a dust free environment. The coverslips were coated with Ilford K2 autoradiographic emulsion and incubated at 4°C for 7 to 14 days in a sealed light proof container. Following the exposure period the slides were processed using photographic developer and fixative, and then the cells were stained using haematoxylin/eosin. When the cells were dry they were coverslipped using a mounting medium. The slides were then assessed for obvious signs of toxicity, reduced numbers of cells and poor labelling.

METHOD OF ANALYSIS:
The coded slides were scored using an automated image analysis system linked to a computer program (Grain). The method used to score the slides was an area counting method. A minimum of three slides for each animal were scored with a maximum of 50 cells per slide giving an accumulative total or 150 cells per animal. The number of silver grains within the nucleus were first observed and recorded as the Nuclear Count (N). Three cytoplasmic areas (each equal to the nuclear area) were also scored to give the Mean Cytplasmic Count (C). A net Nuclear Grain Count (N-C), was then calculated. Cells in 'S'-phase were not scored for grain counts, these were easily recognisable due to the dense 'graining' appearance of the nucleus. Mean values of (N), (C), (N-C) and percentage cells in repair (%R)were calculated. Values of (N-C) are typically in the range of -6 to -2 for vehicle controls, although variations in experimental conditions can give values outside of this range.

Evaluation criteria:
In positive responses at least 20% of all cells assessed should be in repair, ie. undergoing unscheduled DNA synthesis, having a net Nuclear Grain (N-C) value of +5 or greater.
Statistics:
No statistics were performed.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
- Range-finding study: No clinical signs were observed in animals at 1000 and 2000 mg/kg and there were no premature deaths. - UDS study (experiment 1+ experiment 2): no clinical signs and no premature deaths were observed.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals: none


RESULTS OF DEFINITIVE STUDY
There were no premature deaths seen in any of the dose groups, and no clinical signs were observed in any animals in either of the two experiments.

Any other information on results incl. tables

Any other information was available.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
Under the test conditions, ITC 288/S did not induced an increase in the incidence of cells undergoing unscheduled DNA synthesis in isolated rat hepatocytes following in vivo exposure for 2 and 16 hours. Therefore, ITC 288/S was considered to be non-genotoxic.
Executive summary:

In an in vivo liver unscheduled DNA synthesis assay (Durward, 1997), ITC 288/S was administered to male rats at the maximum recommended dose of 2000 mg/kg with 700 mg/kg as the lower dose level. The study was performed in two parts, in experiment 1 the livers were perfused approximately 16 hours after dosing and, in experiment 2, perfusion was performed approximately 2 hours after dosing. Following perfusion the liver hepatocytes were processed to give stained slides which were then scored using a microscope and an automated image analysis system. Further groups of rats were given a single oral dose of distilled water, acetylaminofluorene (AAF) at 16 hours or N,N'-dimethyl hydrazine dihydrochloride (NDHC) at 2 hours to serve as vehicle and positive controls respectively. There was no increase in the incidence of unscheduled DNA synthesis in animals dosed with test material at either time point. The positive controls both produced marked increases in the incidence of cells in repair. Under the conditions of this test, ITC 288/S was considered to be non-genotoxic.

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